Increased osmolarity does not change the protein level of endogenous ubiquitin.

<p>BHK clone 19 myc-CLN3-expressing cells were grown under isotonic (300 mOsm) or hyperosmotic conditions. Osmolarity was increased at 100 mOsm intervals to 500, 600 or 800 mOsm by the addition of NaCl plus urea (1.5∶1 molar ratio). After being exposed to 500, 600 or 800 mOsm for 24 hours, cell lysates were prepared using 1% DDM detergent under non-denaturing conditions. Twenty-µg protein from each sample was loaded on a 16.5% Tris-Tricine polyacrylamide gel and immunoblotted with a mouse monoclonal anti-ubiquitin antibody. Increasing osmolarity did not change the expression level of free ubiqutin (8 kDa) or ubiquitinated proteins (∼70 kDa). Immunoblot representative of 3 biological replicates is shown.